The past 3 years have yielded a wealth of information on the physiological role of adiponectin, an adipocyte-specific secretory protein. Adiponectin has gained widespread acceptance as a valuable indicator of systemic insulin sensitivity. However, the precise mechanism of action that leads to the systemic insulin sensitizing effects observed upon exposure to higher levels of the protein in circulation remains elusive. We propose to examine the biogenesis of the molecule at the level of the adipocyte, look at factors that interact with the protein in circulation and take advantage of a number of genetic mouse models to examine systemic effects of adiponectin. Specifically, we aim to: Specific Aim 1) To determine how adipocytes control the assembly and release of adiponectin complexes. A very significant percentage of adiponectin synthesized does not get exported and is degraded intracellularly. We want to understand whether a specific subpopulation of adiponectin molecules is earmarked for degradation, which (if any) stimuli can re-direct this population towards secretion, and whether the oligomerization state plays a role in this process. Furthermore, we want to determine which chaperones are critically involved in the assembly of the bioactive, high molecular weight form, and which residues in adiponectin play a major role in this process. Specific Aim 2) To examine which (if any) serum factors are associated with adiponectin and whether such serum factors play a critical role in adiponectin function. For this purpose, we have generated a battery of monoclonai antibodies that allow us to immuno-precipitate adiponectin from serum under a number of different conditions to test whether different metabolic states affect cause changes in specific serum factors associated with adiponectin under those conditions. Specific Aim 3) To explore the role of adiponectin systemically, taking advantage of a number of different animal models that we have generated. We have generated mouse models that either lack adiponectin, overexpress adiponectin, secrete adiponectin from liver, express the mutant (Cys39Ser) version of adiponectin that fails to assemble into higher order complexes as well as a novel transgenic mouse model that allows the inducible ablation of adipocytes. Intercrosses between these mouse models will allow us to define the mechanism of action of adiponectin in vivo and shed light on structure/function relationships of this adipokine. It is expected that these studies will contribute fundamental insights towards understanding the role of adiponectin in insulin sensitization in hepatocytes, the secretory pathway of adipocvtes, and interactions with serum factors important for its function.